Creatine is an endogenous nutrient produced naturally by the liver and kidneys in most vertebrates. The uses of creatine are many, including use as a supplement to increase muscle mass and enhance muscle performance as well as in emerging applications in the treatment of various disorders (see, e.g., WO 02/22135) such as, without limitation, Parkinson's disease (Matthews, R. T., et al., (1999) Exp. Neurol., 157:142-149), Huntington's disease, various neuromuscular Disorders (Klivenyi, P., et al. (1999) Nat. Med., 5:347-350), hypoxia and ishemic brain diseases such as stroke (Balestrino, M., et al. (1999) Brain Res., 816:124-130; Dechent, P., et al. (1999) Am. J. Physiol. 277:R698-R704), various muscular dystrophies (Felber, S., et al. (2000) Neurol. Res., 22:145-150; Willer, B., et al. (2000) Rheumatology, 39:293-298; Rawson, E. S. and Clarkson, P. M. (2000) Int. J. Sports Med., 21:71-75), various skin disorders (U.S. patent application Ser. No. 09/852,966) and heart disease (Gordon, A., et al. (1995) Cardiovasc. Res., 30:413-418; Earnest, C. P., et al. (1996) Clin. Sci., 91:113-118). Creatine may also be used as an anti-inflammatory agent (U.S. patent application No. 10/365,666; Khanna, N. K. and Madan, B. R. (1978) Arch. Int. Pharmacodyn. Ther., 231:340-350). Notably, local administration of creatine can be achieved by absorption through the skin (U.S. Pat. No. 6,413,552; WO 96/33707).
Typically, creatine is taken up into muscle cells by specific receptors and converted to phosphocreatine by creatine kinase. Muscle cells, including skeletal muscle and the heart muscle, function by utilizing cellular energy released from the conversion of adenosine triphosphate (ATP) to adenosine diphosphate (ADP). The amount of phosphocreatine in the muscle cell determines the amount of time it will take for the muscle to recover from activity and regenerate ATP. Phosphocreatine is a rapidly accessible source of phosphate required for regeneration of ATP and sustained use of the muscle.
For example, energy used to expand and contract muscles is supplied from ATP. ATP is metabolized in the muscle by cleaving a phosphate radical to release energy needed to contract the muscle. Adenosine diphosphate (ADP) is formed as a byproduct of this metabolism.
The most common sources of ATP are from glycogen and creatine phosphate. Creatine phosphate is favored as a ready source of phosphate because it is able to resynthesize ATP at a greater rate than is typically achieved utilizing glycogen. Therefore, increasing the amount of creatine in the muscle increases the muscle stores of phosphocreatine and has been proven to increase muscle performance and increase muscle mass.
However, creatine itself is poorly soluble in an aqueous solution (about 10-15 mg/ml). Further, creatine is not well absorbed from the gastrointestinal (GI) tract. Indeed, creatine has been estimated to have a 14% or less absorption rate from the GI tract. Creatine also has low oral bioavailability due, in part, to 1) low lipophilicity and therefore poor membrane permeability, and 2) rapid conversion to creatinine in the acidic environment of the stomach (Edgar and Shiver, J. Amer. Chem. Soc., 47:1179-1188, 1925). Thus, current products require administration of large amounts (typically 5 grams or more) of creatine in order to be effective, which causes such side effects as bloating, gastrointestinal (GI) distress, diarrhea, and the like.
These shortcomings and side effects can be avoided by the administration of creatine esters, which are converted into creatine by esterases found in a variety of cells and biological fluids (see, e.g., WO 02/22135). Creatine esters, such as creatine ethyl ester, are more water soluble (over 200 mg/ml) and lipophilic than creatine and therefore have a greater bioavailability. Additionally, the carboxylic acid functional group of creatine is masked through esterification in creatine esters, thereby preventing the formation of the undesired product creatinine.
It is known that creatine esters can be formed via an acid catalyzed reaction between creatine and suitable alcohols, such as methanol, ethanol, and the like.
Current protocols for generating creatine esters employ an external source of hydrogen chloride acid (HCl), such as compressed, caustic HCl gas, in the esterification of anhydrous creatine (see, e.g., Dox and Yoder, J. Biol. Chem., 67:671-673, 1922). The requirement for compressed, caustic HCl gas, however, complicates production of creatine esters and causes the rapid (and ultimately costly) corrosion of metals used in the system and the surrounding area. Additionally, the use of compressed cylinders of HCl gas introduces significant safety risks and renders adding stoichiometric amounts of the acid difficult.
Clearly, a need exists for a process capable of more efficiently and effectively producing creatine esters.